Blood is a liquid tissue that includes red cells, white cells, corpuscles, and platelets dispersed in a liquid phase. The liquid phase is plasma, which includes acids, lipids, dissolved electrolytes, and proteins. One particular protein present in the liquid phase is fibrinogen. When bleeding occurs, fibrinogen reacts with thrombin (an enzyme) to form an insoluble fibrin clot.
In a wide variety of circumstances, animals, including humans, can suffer from bleeding due to wounds or during surgical procedures. In some circumstances, the bleeding is relatively minor, and normal blood clotting functions in addition to the application of simple first aid are all that is required. In other circumstances substantial bleeding can occur. These situations usually require specialized equipment and materials as well as personnel trained to administer appropriate aid.
In an effort to address the above-described problems, materials have been developed for controlling excessive bleeding. Topical Absorbable Hemostats (TAHs) are widely used in surgical applications. TAHs encompass products based on various woven or non-woven fabrics or sponges, typically made of at least partially resorbable materials, ranging from natural to synthetic polymers and combinations thereof, including lactide-glycolide based co-polymers such as polyglactin 910, oxidized cellulose, oxidized regenerated cellulose (ORC), gelatin, collagen, chitin, chitosan, etc. To improve the hemostatic performance, scaffolds based on the above materials can be combined with biologically-derived clotting factors, such as thrombin and/or fibrinogen.
A number of hemostatic formulations currently available on the market or in development utilize lyophilized fibrinogen, frequently in combination with lyophilized thrombin, with hemostatic formulations applied in the form of dry powder, semi-liquid paste, liquid formulation, or optionally disposed on a supporting scaffold such as absorbable fabric scaffold.
A number of prior art references are directed to blood plasma analysis for fibrin or fibrinogen and related assays, development of monoclonal antibodies (mABs), and mABs directed against fibrinogen degradation products. These prior art references teach assays whereby no thrombin (or only the inactive precursor prothrombin) is present in the sample; are not applicable to dry lyophilized proteins of interest; have no solubilization steps and/or thrombin inactivation steps.
An article entitled “Useful laboratory tests for studying thrombogenesis in acute cardiac syndromes”, Fareed et al., Clinical Chemistry. 44(8 Pt 2):1845-53, 1998, discloses utilizing antibodies to fibrinopeptide A (FpA) in ELISA and radioimmunoassay methods for clinical applications, which is a common use for antibodies against FpA as a marker for thrombosis (intravascular blood clots).
An article entitled “Fibrin detected in plasma of patients with disseminated intravascular coagulation by fibrin-specific antibodies consists primarily of high molecular weight factor XIIIa-crosslinked and plasmin-modified complexes partially containing fibrinopeptide A”, Pfitzner et al., Thrombosis & Haemostasis. 78(3):1069-78, 1997, discloses that plasma samples from patients with active clotting were evaluated with various antibodies (including antibody to fibrinopeptide A) to characterize the fibrinogen/fibrin in clot complexes. The starting sample material was plasma.
An article entitled “The conversion of fibrinogen to fibrin: recombinant fibrinogen typifies plasma fibrinogen”, Gorkun et al., Blood. 89(12):4407-14, 1997, discloses that HPLC was used to quantify the amount of FpA released from the two forms of fibrinogen. The starting sample material was liquid (plasma).
An article entitled “Isolation and characterization of the fibrin intermediate arising from cleavage of one fibrinopeptide A from fibrinogen” Shainoff et al., Journal of Biological Chemistry. 271(39):24129-37, 1996, discloses a study of “alpha-profibrin” (fibrin monomer with a single fibrinopeptide A released, rather than the usual two FpA peptides released) and its ability to polymerize. A monoclonal antibody that was directed against FpA was used to determine if FpA had been cleaved. The starting material was a solution containing fibrinogen, fibrin or intermediates.
An article entitled “A monoclonal antibody, specific for human fibrinogen, fibrinopeptide A-containing fragments and not reacting with free fibrinopeptide” Koppert et al., Blood. 66(3):503-7, 1985, describes the production of a monoclonal antibody to FpA.
An article entitled “Monoclonal antibodies to different neo-epitopes on fibrinogen and fibrin degradation products”, Amiral et al., Blood Coagul Fibrinolysis. 1990 October; 1(4-5):447-52, discloses an attempt to develop various monoclonal antibodies specific for the neo-epitopes unmasked during the degradation of fibrin or fibrinogen that are classified in three reactivity classes: D and D-dimer, D-dimer and early fibrinogen, and fibrin degradation products. These monoclonal antibodies were used to develop latex slide assays and ELISA techniques. Two types of citrated plasma assays were obtained; those which were specific for fibrin-related products and those evaluating the totality of fibrin or fibrinogen degradation products.
An article entitled “A monoclonal antibody-based quantitative enzyme immunoassay for the determination of plasma fibrinogen concentrations”, Hoegee-de Nobel et al., Thromb Haemost. 1988 Dec. 22; 60(3):415-8, discloses monoclonal antibody-based quantitative enzyme immunoassay for the determination of plasma fibrinogen concentrations and teaches that immunological assays for fibrinogen fibrinopeptide A, which do not detect degradation products of fibrinogen.
An article entitled “Comparison of several mouse and rat monoclonal antibodies against human fibrinogen”, Marecek et al., Hybridoma. 1996 December; 15(6):423-7, discloses that six monoclonal antibodies raised against human fibrinogen have been characterized. Mouse monoclonal antibodies were targeted against sequential epitopes on the immunodominant D-domain of fibrinogen and they crossreacted with all molecules containing the D-domain [fibrin, fibrin(ogen)-degradation products].
U.S. Pat. No. 7,790,410 B2 discloses a method of screening a candidate material for hemocompatibility comprising: (1) contacting a candidate material with fibrinogen; (2) contacting the candidate material from step (1) with thrombin; determining a presence or level of a fibrinogen cleavage product from step (2); and determining if the candidate material is hemocompatible based on the presence or level of the fibrinogen cleavage product.
U.S. Pat. No. 6,074,837 discloses a competitive immunoassay for determining fibrin and fibrin degradation products in a sample, said immunoassay comprising individually contacting said sample and a positive control of known fibrin and fibrin degradation products concentration with a labeled antibody which specifically binds to said fibrin and fibrin degradation products, and after a suitable incubation period, separating bound labeled antibody from unbound labeled antibody, measuring bound label, and comparing measured bound label in the contacted sample to measured bound label in the contacted positive control to determine the presence or amount of said fibrin and fibrin degradation products in said sample, wherein the improvement comprises using a modified fibrinogen as said fibrin and fibrin degradation products in said positive control. The reference further discloses a sandwich immunoassay for determining fibrin and fibrin degradation products in a sample, said immunoassay comprising coating wells of a microtiter plate with a first antibody which specifically binds to said fibrin, said fibrin degradation products and fibrin monomer; contacting said sample and a positive control of known fibrin or fibrin monomer concentration to separate wells of said microtiter plate; removing any unbound sample or positive control from its respective well; contacting each well with a labeled anti-first antibody antibody; measuring any bound label in each well and comparing the measured bound label in the sample wells to the measured bound label in the positive control wells to determine the presence or amount of said fibrin and fibrin degradation products in the sample, wherein the improvement comprises using a modified fibrinogen as said fibrin or fibrin monomer in said positive control, the modified fibrinogen obtained by a process comprising the following steps: (1) partially reducing 3 to 25 micromolar fibrinogen with 0.25 millimole per nanomole of said fibrinogen with a reducing agent at 30-40° C. under non-denaturing conditions in the absence of divalent cations for 0.5 to 1.5 hours, then (2) blocking thiol groups of any free cysteines formed during step (1) by reacting the product of step (1) with a blocking agent that does not cause precipitation of the product of step (2), then (3) reacting the product of step (2) with a clotting enzyme in a physiological buffer in the absence of divalent cations to release fibrinopeptides A and B, and (4) terminating the activity of said clotting enzyme.
U.S. Pat. No. 5,876,947 discloses a continuous cell line, identified as P10 and deposited as ATCC Accession No. HB-12398 that produces a monoclonal antibody that binds specifically with an epitope defined by a specific amino acid sequence and further discloses a monospecific antibody or fragment thereof that binds specifically to the epitope as present in fibrinogen, fibrinopeptide B, or des-Arg fibrinopeptide B.
U.S. Pat. No. 4,438,209 discloses a competitive radioimmunoassay method for determining the concentration of fibrinopeptide A in plasma wherein first, a sample of blood is collected, the thrombin in said sample is inhibited by an inhibiting amount of a thrombin inhibitor and plasma is separated from said sample, and second, a sample of said plasma is contacted under radioimmunoassay competitive binding conditions with a sufficient amount of an antibody to fibrinopeptide A and radioactively labeled fibrinopeptide A, thereafter, antibody bound fibrinopeptide A is separated from the unbound fibrinopeptide A and radioactivity measured, the improvement comprising using as the inhibitor for thrombin an inhibitor selected from the group consisting of D-phenylalanyl-L-propyl-L-N-[2(1-chloro-7-guanidoheptane-2-one)] the hydrochloric acid addition salt thereof; the hydrofluoric acid addition salt thereof, the acetic acid addition salt thereof and the citric acid addition salt thereof.
European Patent publication EP 345,811 A2 discloses a hybridoma which secretes a monoclonal antibody specific for human fibrinopeptide A, but which does not react with either intact fibrinogen or human fibrinopeptide A-containing fibrinogen fragments. The reference further discloses a method for determining free FpA comprising (a) immobilizing a monoclonal antibody, said monoclonal antibody being according to claim 1, (b) contacting the immobilized monoclonal antibody from (a) with labeled hFPA peptide or a labeled fragment thereof or a Tyr derivative thereof, and a plasma sample, and (c) assaying for the label.
U.S. Pat. No. 5,817,768 discloses a monospecific antibody, which binds with an epitope of the αE subunit of fibrinogen, wherein said monospecific antibody is produced by a hydridoma cell line selected from the group consisting of hybridoma cell line identified as #3-10, hybridoma cell line identified as #29-1, and hybridoma cell line #148-B.
PCT patent publication WO2007/030571 A2 discloses identification of disease target molecules and the development of imaging reagents and diagnostic assays specific to those molecules. Described therein are methods and reagents for the identification of molecular targets specific to a disease or disease state, methods of imaging technology which can be used, the development of specific molecular imaging reagents, clinical validation of the imaging reagents, and clinical indications for molecular imaging. The reference claims a target-specific imaging reagent, comprising an affinity agent coupled to an imaging agent, wherein said affinity agent specifically binds to a biological molecule, wherein expression of said biological molecule is predictive of a disease or a disease state.
PCT patent publication WO 2007/030531 describes both the identification of disease target molecules and the development of imaging reagents and diagnostic assays specific to those molecules. Described therein are methods and reagents for the identification of molecular targets specific to a disease or disease state, methods of imaging technology which can be used, the development of specific molecular imaging reagents, clinical validation of the imaging reagents, and clinical indications for molecular imaging. The reference claims a target-specific imaging reagent, comprising an antibody coupled to an imaging agent detectable by magnetic resonance, wherein said antibody specifically binds to Glypican-3, wherein expression of Glypican-3 is predictive of liver cancer.
U.S. Patent application publication No. 20110053193 discloses a method for determining the activity or functionality of either a first reactive component or a second reactive component in an unreacted admixture of the first reactive component and the second reactive component, comprising the steps of (a) reversibly inhibiting the first reactive component to yield a mixture having an inactivated first reactive component and the second reactive component; (b) adding to the mixture a known amount of the second reactive component when evaluating the activity of the first reactive component, or a known amount of the first reactive component when evaluating the activity of the second reactive component; (c) reversibly activating the first reactive component; (d) allowing the first reactive component to react with the second reactive component originally present in the admixture and the known amount of the second reactive component, or allowing the first reactive component to react with the second reactive component originally present in the admixture and the known amount of the first reactive component; and (e) determining the activity or functionality of first or second reactive component originally present in the admixture.
Hemostatic patches or pads containing lyophilized thrombin and fibrinogen, optionally on absorbable scaffolds, lack an assay to quantify the activation status of the biological components (i.e. whether fibrinogen has been converted to fibrin). Because fibrinogen and thrombin spontaneously react upon hydration, exposure of the hemostatic pad to moisture could cause premature activation (prior to application to the bleeding site) of the biological components and could potentially impact the pad's overall stability and performance. Currently fibrin sealant delivery devices, deploying a mixture of fibrinogen and thrombin solutions on a wound for hemostatic or tissue sealing applications lack a test capable of directly evaluating fibrinogen to fibrin conversion. One objective of the Applicants' work was to develop a robust assay that measures both the rate and extent of the fibrinogen to fibrin conversion reaction as well as to measure the amount of intact fibrinogen in the presence of thrombin.